Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

ABSTRACT

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation—when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. Subsequent exposure to excitation energy will excite the semiconductor nanocrystal in he probe, causing the emission of electromagnetic radiation. Further described are processes for respectively: making the semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/978,450 filed Nov. 25, 1997, and now issued as U.S. Pat. No.5,990,479 on Nov. 23, 1999.

The invention described herein arose in the course of, or under,Contract No. DE-AC03-SF00098 between the United States Department ofEnergy and the University of California for the operation of the ErnestOrlando Lawrence Berkeley National Laboratory. The Government may haverights to the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to organo luminescent semiconductor nanocrystalprobes for biological applications wherein the probes includes aplurality of semiconductor nanocrystals capable of luminescence and/orabsorption and/or scattering or diffraction when excited by a radiationor particle beam.

2. Description of the Related Art

Fluorescent labeling of biological systems is a well known analyticaltool used in modem biotechnology as well as analytical chemistry.Applications for such fluorescent labeling include technologies such asmedical (and non-medical) fluorescence microscopy, histology, flowcytometry, fluorescence in-situ hybridization (medical assays andresearch), DNA sequencing, immuno-assays, binding assays, separation,etc.

Conventionally, such fluorescent labeling involves the use of an organicdye molecule bonded to a moiety which, in turn, selectively bonds to aparticular biological system, the presence of which is then identifiedby excitation of the dye molecule to cause it to fluoresce. There are anumber of problems with such an analytical system. In the first place,the emission of light of visible wavelengths from an excited dyemolecule usually is characterized by the presence of a broad emissionspectrum as well as a broad tail of emissions on the red side of thespectrum, i.e., the entire emission spectrum is rather broad. As aresult, there is a severe limitation on the number of different colororganic dye molecules which may be utilized simultaneously orsequentially in an analysis since it is difficult to eithersimultaneously or even non-simultaneously detect or discriminate betweenthe presence of a number of different detectable substances due to thebroad spectrum emissions and emission tails of the labelling molecules.Another problem is that most dye molecules have a relatively narrowabsorption spectrum, thus requiring either multiple excitation beamsused either in tandem or sequentially for multiple wavelength probes, orelse a broad spectrum excitation source which is sequentially used withdifferent filters for sequential excitation of a series of probesrespectively excited at different wavelengths.

Another problem frequently encountered with existing dye molecule labelsis that of photostability. Available fluorescent molecules bleach, orirreversibly cease to emit light, under repeated excitation (10⁴-10⁸)cycles of absorption/emission. These problems are often surmounted byminimizing the amount of time that the sample is exposed to light, andby removing oxygen and/or other radical species from the sample.

In addition, the probe tools used for the study of these systems byelectron microscopy techniques are completely different from the probesused for study by fluorescence. Thus, it is not possible to label amaterial with a single type of probe for both electron microscopy andfor fluorescence.

It would, therefore, be desirable to provide a stable probe material forbiological applications having a wide absorption band and capable ofexhibiting either a detectable change in absorption or of emittingradiation in a narrow wavelength band, without the presence of the largered emission tails characteristic of dye molecules (thereby permittingthe simultaneous use of a number of such probe materials, each emittinglight of a different narrow wavelength band) and/or capable ofscattering or diffracting radiation. It would also be equally desirableto provide a single, stable probe material which can be used to imagethe same sample by both light and electron microscopy.

SUMMARY OF THE INVENTION

The invention comprises a luminescent semiconductor nanocrystal compoundcapable of linking to an affinity molecule to form an organo luminescentsemiconductor nanocrystal probe capable of luminescence and/orabsorption and/or scattering or diffracting when excited by anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam, and capable of exhibiting a detectable change inabsorption and/or of emitting radiation in a narrow wavelength bandand/or scattering or diffracting when so excited. The luminescentsemiconductor nanocrystal compound preferably comprises: (1) asemiconductor nanocrystal capable of luminescence and/or absorptionand/or scattering or diffraction when excited by an electromagneticradiation source (of broad or narrow bandwidth) or a particle beam, andcapable of exhibiting a detectable change in absorption and/or ofemitting radiation in a narrow wavelength band and/or scattering ordiffracting when excited; and (2) a linking agent having a first portionlinked to the semiconductor nanocrystal, and a second portion capable oflinking to an affinity molecule.

The invention further comprises an organo luminescent semiconductornanocrystal probe formed by linking the above described luminescentsemiconductor nanocrystal compound to an affinity molecule capable ofbonding to a detectable substance in a material. As a result the organoluminescent semiconductor nanocrystal probe, in one embodiment, iscapable of absorbing or scattering or diffracting energy from either aparticle beam or an electromagnetic radiation source (of broad or narrowbandwidth), and is capable of emitting electromagnetic radiation in anarrow wavelength band when so excited; while in another embodiment theamount of energy so absorbed, or scattered, or diffracted from either aparticle beam or an electromagnetic radiation source (of broad or narrowbandwidth), is detectable, i.e., the change in absorption, scattering,ordiffraction is detectable.

Therefore, treatment of a material with the organo luminescentsemiconductor nanocrystal probe, and subsequent exposure of this treatedmaterial to excitation energy (from either a particle beam or anelectromagnetic radiation source of broad or narrow bandwidth) todetermine the presence of the detectable substance within the material,will excite the semiconductor nanocrystals in the organo luminescentsemiconductor nanocrystal probe bonded to the detectable substance,resulting in the emission of electromagnetic radiation of a narrowwavelength band and/or a detectable change in the amount of energy beingabsorbed and/or scattered or diffracted, signifying the presence, in thematerial, of the detectable substance bonded to the organo luminescentsemiconductor nanocrystal probe.

The invention also comprises a process for making the luminescentsemiconductor nanocrystal compound and for making the organo luminescentsemiconductor nanocrystal probe comprising the luminescent semiconductornanocrystal compound linked to an affinity molecule capable of bondingto a detectable substance. The organo luminescent semiconductornanocrystal probe of the invention is stable with respect to repeatedexcitation by light, or exposure to oxygen or other radicals. Theinvention further comprises a process for treating a material, such as abiological material, to determine the presence of a detectable substancein the material which comprises contacting the material with the organoluminescent semiconductor nanocrystal probe, removing from the materialportions of the organo luminescent semiconductor nanocrystal probe notbonded to the detectable substance, and then exposing the material toactivation energy from either an electromagnetic radiation source (ofbroad or narrow bandwidth) or a particle beam. The presence of thedetectable substance in the material is then determined either bymeasuring the absorption of energy by the organo luminescentsemiconductor nanocrystal probe and/or detecting the emission ofradiation of a narrow wavelength band by the organo luminescentsemiconductor nanocrystal probe and/or detecting the scattering ordiffraction by the organo luminescent semiconductor nanocrystal probe,indicative (in either case) of the presence of the organo luminescentsemiconductor nanocrystal probe bonded to the detectable substance inthe material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of the luminescent semiconductor nanocrystalcompound of the invention.

FIG. 2 is a block diagram of the organo luminescent semiconductornanocrystal probe of the invention.

FIG. 3 is a block diagram showing the affinity between a detectablesubstance and the organo luminescent semiconductor nanocrystal probe ofthe invention.

FIG. 4 is a flow sheet illustrating the process of forming the organoluminescent semiconductor nanocrystal probe of the invention.

FIG. 5 is a flow sheet illustrating a typical use of the organoluminescent semiconductor nanocrystal probe of the invention indetecting the presence of a detectable substance in a material such as abiological material.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises a luminescent semiconductor nanocrystal compoundcapable of linking to an organic molecule and capable of exhibiting adetectable change in absorption and/or of emitting electromagneticradiation in a narrow wavelength band and/or scattering or diffractingwhen excited by either an electromagnetic radiation source (of broad ornarrow bandwidth) or a particle beam. The luminescent semiconductornanocrystal compound, in turn, comprises: (1) semiconductor nanocrystalscapable of exhibiting a detectable change in absorption and/or ofemitting electromagnetic radiation in a narrow wavelength band whenexcited by either an electromagnetic radiation source (of broad ornarrow bandwidth) or a particle beam; and (2) one or more linking agentseach having a first portion linked to the semiconductor nanocrystal anda second portion capable of linking to an organic affinity molecule.

The invention also comprises the above described luminescentsemiconductor nanocrystal compound linked to the organic affinitymolecule (through the linking agent) to form an organo luminescentsemiconductor nanocrystal probe capable of bonding to a detectablesubstance and capable of exhibiting a detectable change in absorptionand/or of emitting electromagnetic radiation in a narrow wavelength bandand/or scattering or diffracting when excited by either anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam. Treatment of a material (typically a biological material)with the organo luminescent semiconductor nanocrystal probe, andsubsequent exposure of this treated material to excitation energy, asdescribed above, to determine the presence of the detectable substancewithin the material, will excite the semiconductor nanocrystal in theorgano luminescent semiconductor nanocrystal probe bonded to thedetectable substance, causing the detectable change in absorption and/oremission of electromagnetic radiation of a narrow wavelength band and/orscattering or diffraction signifying (in either instance) the presencein the material, of the detectable substance bonded to the organoluminescent semiconductor nanocrystal probe.

The invention also comprises a process for making the luminescentsemiconductor nanocrystal compound, and a process for making the organoluminescent semiconductor nanocrystal probe comprising the luminescentsemiconductor nanocrystal compound linked to an affinity moleculecapable of bonding to a detectable substance.

The invention further comprises a process for treating a material, suchas a biological material, to determine the presence of a detectablesubstance in the material which comprises: (1) contacting the materialwith the organo luminescent semiconductor nanocrystal probe, (2)removing from the material portions of the organo luminescentsemiconductor nanocrystal probe not bonded to the detectable substance,(3) exposing the material to energy (such as the above-describedelectromagnetic energy source or particle beam) capable of exciting thesemiconductor nanocrystal to cause a detectable change in absorptionand/or emission of electromagnetic radiation of a narrow wavelength bandand/or scattering or diffraction signifying (in either instance) thepresence of the organo luminescent semiconductor nanocrystal probebonded to the detectable substance in the material, and (4) detectingeither the change in absorbed energy or the electromagnetic radiationemitted or the scattering or diffraction by the semiconductornanocrystal in the organo luminescent semiconductor nanocrystal probe.

a. Definitions

By use of the terms “nanometer crystal” or “nanocrystal” herein is meantan organic or inorganic single crystal particle having an averagecross-section no larger than about 20 nanometers (nm) or 20×10⁻⁹ meters(200 Angstroms), preferably no larger than about 10 nm (100 Angstroms)and a minimum average cross-section of about 1 nm, although in someinstances a smaller average cross-section nanocrystal, i.e., down toabout 0.5 nm (5 Angstroms), may be acceptable. Typically the nanocrystalwill have an average cross-section ranging in size from about 1 nm (10Angstroms) to about 10 nm (100 angstroms).

By use of the term “semiconductor nanocrystal” is meant a nanometercrystal or nanocrystal of Group II-VI and Group III-V semiconductorcompounds capable of emitting electromagnetic radiation upon excitation,although the use of Group IV semiconductors such as germanium orsilicon, or the use of organic semiconductors, may be feasible undercertain conditions.

By use of the term “a narrow wavelength band”, with regard to theelectromagnetic radiation emission of the semiconductor nanocrystal, ismeant a wavelength band of emissions not exceeding about 40 nm, andpreferably not exceeding about 20 mn in width and symmetric about thecenter, in contrast to the emission bandwidth of about 100 nm for atypical dye molecule, with a red tail which may extend the band widthout as much as another 100 nm. It should be noted that the bandwidthsreferred to are determined from measurement of the width of theemissions at half peak height (FWHM), and are appropriate in the rangeof 200 nm to 2000 nm.

By use of the term “a broad absorption band”, with regard to theelectromagnetic radiation absorption of the semiconductor nanocrystal ismeant a continuously increasing absorption from the onset, which occursnear to, but at slightly higher energy than the “narrow wavelength band”of the emission. This is in contrast to the “narrow absorption band” ofdye molecules which occurs near the emission peak on the high energyside, but drops off rapidly away from that wavelength.

By use of the term “detectable substance” is meant an entity or group,the presence or absence of which in a material such as a biologicalmaterial, is to be ascertained by use of the organo-luminescentsemiconductor nanocrystal probe of the invention.

By use of the term “affinity molecule” is meant the portion of theorgano luminescent semiconductor nanocrystal probe of the inventionwhich will selectively bond to a detectable substance (if present) inthe material (e.g., biological material) being analyzed.

By use of the term “linking agent” is meant a substance capable oflinking with a semiconductor nanocrystal and also capable of linking toan affinity molecule.

The terms “link” and “linking” are meant to describe the adherencebetween the affinity molecule and the semiconductor nanocrystals, eitherdirectly or through a moiety identified herein as a linking agent. Theadherence may comprise any sort of bond, including, but not limited to,covalent, ionic, hydrogen bonding, Van der Waals' forces, or mechanicalbonding, etc.

The terms “bond” and “bonding” are meant to describe the adherencebetween the affinity molecule and the detectable substance. Theadherence may comprise any sort of bond, including, but not limited to,covalent, ionic, or hydrogen bonding, Van der Waals' forces, ormechanical bonding, etc.

The term “luminescent semiconductor nanocrystal compound”, as usedherein, is intended to defme a semiconductor nanocrystal linked to oneor more linking agents and capable of linking to an affinity molecule,while the term “organo-luminescent semiconductor nanocrystal probe” isintended to define a luminescent semiconductor nanocrystal compoundlinked to an affinity molecule.

The term “glass” as used herein is intended to include one or moreoxides of silicon, boron, and/or phosphorus, or a mixture thereof, aswell as the further optional inclusion of one or more metal silicates,metal borates or metal phosphates therein.

b. The Semiconductor Nanocrystals

The semiconductor nanocrystals useful in the practice of the inventioninclude nanocrystals of Group II-VI semiconductors such as MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe; and nanocrystals of GroupIII-V semiconductors such as GaAs, InGaAs, InP, and InAs. As mentionedabove, the use of Group IV semiconductors such as germanium or silicon,or the use of organic semiconductors, may also be feasible under certainconditions.

Formation of nanometer crystals of Group III-V semiconductors isdescribed in copending and commonly assigned Alivisatos et al. U.S. Pat.No. 5,751,018; Alivisatos et al. U.S. Pat. No. 5,505,928; and Alivisatoset al. U.S. Pat. No. 5,262,357, which also describes the formation ofGroup II-VI semiconductor nanocrystals, and which is also assigned tothe assignee of this invention. Also described therein is the control ofthe size of the semiconductor nanocrystals during formation usingcrystal growth terminators. The teachings of Alivisatos et al. U.S. Pat.No. 5,751,018, and Alivisatos et al. U.S. Pat. No. 5,262,357 are eachhereby specifically incorporated by reference.

In a preferred embodiment, the nanocrystals are used in a core/shellconfiguration wherein a first semiconductor nanocrystal forms a coreranging in diameter, for example, from about 20 Å to about 100 Å, with ashell of another semiconductor nanocrystal material grown over the corenanocrystal to a thickness of, for example, 1-10 monolayers inthickness. When, for example, a 1-10 monolayer thick shell of CdS isepitaxially grown over a core of CdSe, there is a dramatic increase inthe room temperature photoluminescence quantum yield. Formation of suchcore/shell nanocrystals is described more fully in a publication by oneof us with others entitled “Epitaxial Growth of Highly LuminescentCdSe/CdS Core/Shell Nanocrystals with Photostability and ElectronicAccessibility”, by Peng, Schlamp, Kadavanich, and Alivisatos, publishedin the Journal of the American Chemical Society, Volume 119, No. 30.1997, at pages 7019-7029, the subject matter of which is herebyspecifically incorporated herein by reference.

The semiconductor nanocrystals used in the invention will have acapability of emitting light within a narrow wavelength band of about 40nm or less, preferably about 20 nm or less, thus permitting thesimultaneous use of a plurality of differently colored organoluminescent semiconductor nanocrystal probes with differentsemiconductor nanocrystals without overlap (or with a small amount ofoverlap) in wavelengths of emitted light (unlike the use of dyemolecules with broad emission lines (e.g., ˜100 nm) and broad tails ofemission (e.g., another 100 nm) on the red side of the spectrum), thusallowing for the simultaneous detection of a plurality of detectablesubstances.

c. Affinity Molecule

The particular affinity molecule forming a part of theorgano-luminescent semiconductor nanocrystal probe of the invention willbe selected based on its affinity for the particular detectablesubstance whose presence or absence, for example, in a biologicalmaterial, is to be ascertained. Basically, the affinity molecule maycomprise any molecule capable of being linked to a luminescentsemiconductor nanocrystal compound which is also capable of specificrecognition of a particular detectable substance. In general, anyaffinity molecule useful in the prior art in combination with a dyemolecule to provide specific recognition of a detectable substance willfind utility in the formation of the organo-luminescent semiconductornanocrystal probes of the invention. Such affinity molecules include, byway of example only, such classes of substances as monoclonal andpolyclonal antibodies, nucleic acids (both monomeric and oligomeric),proteins, polysaccharides, and small molecules such as sugars, peptides,drugs, and ligands. Lists of such affinity molecules are available inthe published literature such as, by way of example, the “Handbook ofFluorescent Probes and Research Chemicals”, (sixth edition) by R. P.Haugland, available from Molecular Probes, Inc.

d. The Linking Agent

The organo-luminescent semiconductor nanocrystal probe of the inventionwill usually find utility with respect to the detection of one or moredetectable substances in organic materials, and in particular to thedetection of one or more detectable substances in biological materials.This requires the presence, in the organo-luminescent semiconductornanocrystal probe, of an affinity molecule or moiety, as describedabove, which will bond the organo-luminescent semiconductor nanocrystalprobe to the detectable substance in the organic/biological material sothat the presence of the detectable material may be subsequentlyascertained. However, since the semiconductor nanocrystals areinorganic, they may not bond directly to the organic affinity molecule.In these case therefore, there must be some type of linking agentpresent in the organo-luminescent semiconductor nanocrystal probe whichis capable of forming a link to the inorganic semiconductor nanocrystalas well as to the organic affinity molecule in the organo-luminescentsemiconductor nanocrystal probe.

One form in which the semiconductor nanocrystal may be linked to anaffinity molecule via a linking agent is by coating the semiconductornanocrystal with a thin layer of glass, such as silica (SiO_(x) wherex=1-2), using a linking agent such as a substituted silane, e.g.,3-mercaptopropyl-trimethoxy silane to link the nanocrystal to the glass.The glass-coated semiconductor nanocrystal may then be further treatedwith a linking agent, e.g., an amine such as3-aminopropyl-trimethoxysilane, which will function to link theglass-coated semiconductor nanocrystal to the affinity molecule. Thatis, the glass-coated semiconductor nanocrystal may then be linked to theaffinity molecule. It is within the contemplation of this invention thatthe original luminescent semiconductor nanocrystal compound may also bechemically modified after it has been made in order to link effectivelyto the affinity molecule. A variety of references summarize the standardclasses of chemistry which may be used to this end, in particular the“Handbook of Fluorescent Probes and Research Chemicals”, (6th edition)by R. P. Haugland, available from Molecular Probes, Inc., and the book“Bioconjugate Techniques”, by Greg Hermanson, available from AcademicPress, New York.

When the semiconductor nanocrystal is coated with a thin layer of glass,the glass, by way of example, may comprise a silica glass (SiO_(x) wherex=1-2), having a thickness ranging from about 0.5 nm to about 10 nm, andpreferably from about 0.5 nm to about 2 nm.

The semiconductor nanocrystal is coated with the coating of thin glass,such as silica, by first coating the nanocrystals with a surfactant suchas tris-octyl-phosphine oxide, and then dissolving the surfactant-coatednanocrystals in a basic methanol solution of a linking agent, such as3-mercaptopropyl-tri-methoxy silane, followed by partial hydrolysiswhich is followed by addition of a glass-affinity molecule linking agentsuch as amino-propyl trimethoxysilane which will link to the glass andserve to form a link with the affinity molecule. When the linking agentdoes not involve the use of a glass coating on the semiconductornanocrystal, it may comprise a number of different materials, dependingupon the particular affinity molecule, which, in turn, depends upon thetype of detectable material being analyzed for. It should also be notedthat while an individual linking agent may be used to link to anindividual semiconductor nanocrystal, it is also within thecontemplation of the invention that more than one linking agent may bondto the same semiconductor nanocrystal and vice versa.

A few examples of the types of linking agents which may be used to linkto both the semiconductor nanocrystal (or to a glass coating on thenanocrystal) and to the organic affinity molecule in the probe areillustrated in the table below, it being understood that this is notintended to be an exhaustive list:

Linking Agent Structure Name

N-(3-aminopropyl)3-mercapto-benzamide

3-aminopropyl-trimethoxysilane

3-mercaptopropyl-trimethoxysilane

3-maleimidopropyl-trimethoxysilane

3-hydrazidopropyl-trimethoxysilane

It should be further noted that a plurality of polymerizable linkingagents may be used together to form an encapsulating net or linkagearound an individual nanocrystal (or group of nanocrystals). This is ofparticular interest where the particular linking agent is incapable offorming a strong bond with the nanocrystal. Examples of linking agentscapable of bonding together in such a manner to surround the nanocrystalwith a network of linking agents include, but are not limited to:diacetylenes, acrylates, acrylamides, vinyl, styryl, and theaforementioned silicon oxide, boron oxide, phosphorus oxide, silicates,borates and phosphates.

e. The Excitation of the Probe and Detection of Emission/Absorption

As previously mentioned, the organo luminescent semiconductornanocrystal probe of the invention is capable of being excited over abroad bandwidth, yet exhibits emission in a narrow wavelength band, incontrast to the dye molecules used in the prior art. Thuselectromagnetic radiation of wavelength ranging from x-ray toultraviolet to visible to infrared waves may be used to excite theluminescent semiconductor nanocrystals in the probe. In addition, theluminescent semiconductor nanocrystals are capable of excitation frombombardment with a particle beam such as an electron beam (e-beam).Furthermore, because of the broad bandwidth at which the luminescentsemiconductor nanocrystals are excitable, one may use a commonexcitation source for the simultaneous excitation of several probes,i.e., several probes which give off radiation at different frequencies,thus permitting simultaneous excitation and detection of the presence ofseveral probes indicating, for example, the presence of severaldetectable substances in the material being examined.

Thus, for example, a laser radiation source of a given frequency, e.g.,blue light, may be used to excite a first organo luminescentsemiconductor nanocrystal probe capable of emitting radiation of asecond frequency, e.g., red light, indicating the presence, in thematerial being illuminated, of a first detectable substance to which theparticular red light-emitting organo luminescent semiconductornanocrystal probe has bonded. At the same time, the same blue lightlaser source may also be exciting a second organo luminescentsemiconductor nanocrystal probe (in the same material) capable ofemitting radiation of a third frequency, e.g., green light, indicatingthe presence, in the material being illuminated, of a second detectablesubstance to which the particular green light-emitting organoluminescent semiconductor nanocrystal probe has bonded. Thus, unlike theprior art, multiple excitation sources need not be used (because of thebroad bandwidth in which the organo luminescent semiconductornanocrystal probe of the invention is capable of being excited), and thenarrow band of emission of the specific semiconductor nanocrystals ineach probe makes possible the elimination of sequencing and/or elaboratefiltering to detect the emitted radiation.

With respect to the absorption of energy by the probe of the invention,when the excitation source is an electron beam, or an X-ray source, thepresence of the organo luminescent semiconductor nanocrystal probebonded to the detectable substance of interest in the material beinganalyzed can be ascertained using a commercially available energyabsorption or scattering or diffraction detection system wherein changesin absorption or scattering cross section or in diffraction of thematerial being analyzed can be detected, signifying the presence of theprobe in the material, which, in turn, indicates the presence of thedetectable substance to which the probe is bonded in the material beinganalyzed. In addition, it may be possible to use electron or X-raysources to detect the presence of the organo luminescent semiconductornanocrystal probe bonded to the detectable substance by using aconventional detection system for the emission of visible light toobserve the visible emission in the narrow wavelength of emission of theprobe.

The following examples will serve to further illustrate the formation ofthe organo luminescent semiconductor nanocrystal probes of theinvention, as well as their use in detecting the presence of adetectable substance in a material such as a biological material.

Example 1

To illustrate the formation of the luminescent semiconductor nanocrystalcompound (comprising the semiconductor nanocrystals linked to a linkingagent) 20 ml. of a 5 mM solution of (4-mercapto)benzoic acid wasprepared with a pH of 10 using (CH₃)₄NOH.5H₂O. 20 mg oftris-octylphosphine oxide coated CdSe/CdS core/shell nanocrystals wereadded to the solution and stirred until completely dissolved. Theresultant nanocrystal/linking agent solution was heated for 5 hours at50-60° C. and then concentrated to a few ml by evaporation. Then anequal volume of acetone was added and the nanocrystals precipitate outof solution homogeneously. The precipitate was then washed with acetone,dried, and then can be stored.

The luminescent semiconductor nanocrystal compound prepared above can belinked with an appropriate affinity molecule to form the organoluminescent semiconductor nanocrystal probe of the invention to treat abiological material to determine the presence or absence of a detectablesubstance. That is, the luminescent semiconductor nanocrystal compoundprepared above can be linked, for example, with avidin or streptavidin(as the affinity molecule) to form an organo luminescent semiconductornanocrystal probe to treat a biological material to ascertain thepresence of biotin; or the luminescent semiconductor nanocrystalcompound prepared above can be linked with anti-digoxiginen to form anorgano luminescent semiconductor nanocrystal probe to treat a biologicalmaterial to ascertain the presence of digoxiginen.

Example 2

To illustrate the formation of luminescent semiconductor nanocrystalcompound (comprising glass-coated semiconductor nanocrystals linked to alinking agent), 50 μl of 3-mercaptopropyl-trimethoxy silane was added to40 ml of an anhydrous solution of 25 vol. % dimethylsulfoxide inmethanol, and the pH was adjusted to 10-11 using (CH₃)₄NOH.5H₂O. 10 mgof tris-octylphosphine oxide coated CdSe/CdS core-shell particles,prepared by the technique described in the aforementioned Peng, Schlamp,Kadavanich, and Alivisatos article, were then dissolved in thissolution, and stirred for several hours. The solution was diluted with40 ml of methanol adjusted to a pH of 10 with (CH₃)₄NOH.5H₂O, and heatedfor 1 hour at 69° C. The solution was stirred for an hour, and 40 ml ofa 90 vol. % methanol/9.89 vol. % H₂O/0.1 vol. % trimethoxysilylpropylurea/0.01 vol. % aminopropyl-trimethoxy silane solution which had beenstirring for at least an hour, was added, and stirred for 2 hours.Subsequently the reaction was heated to 69° C. for 15 minutes, and thencooled. 10 ml of a 10 vol. % chlorotrimethyl silane solution in methanolwhich had been adjusted to a pH of 10 using (CH₃)₄NOH.5H₂O was added,stirred for 2 hours, then heated to 60° C., and then partiallyconcentrated under vacuum. Once the methanol had all evaporated, thesolution was precipitated with acetone as an oil product comprising theluminescent semiconductor nanocrystal compound. The luminescentsemiconductor nanocrystal compound may then be redissolved in water, andin a variety of buffer solutions to prepare it for linking it to anaffinity molecule to form the organo luminescent semiconductornanocrystal probe of the invention to treat a biological material todetermine the presence or absence of a detectable substance.

Thus, the invention provides an organo luminescent semiconductornanocrystal probe containing a semiconductor nanocrystal capable, uponexcitation by either electromagnetic radiation (of either narrow orbroad bandwidth) or particle beam, of emitting electromagnetic radiationin a narrow wavelength band and/or absorbing energy and/or scattering ordiffracting said excitation, thus permitting the simultaneous usage of anumber of such probes emitting different wavelengths of electromagneticradiation to thereby permit simultaneous detection of the presence of anumber of detectable substances in a given material. The probe materialis stable in the presence of light or oxygen, capable of being excitedby energy over a wide spectrum, and has a narrow band of emission,resulting in an improved material and process for the simultaneousand/or sequential detection of a number of detectable substances in amaterial such as a biological material.

Having thus described the invention what is claimed is:
 1. A semiconductor nanocrystal compound, comprising: a) a water-soluble semiconductor nanocrystal comprising: i) a core comprising a first semiconductor material; and ii) a core-overcoating shell comprising a second semiconductor material; and b) a linking agent linked to said water-soluble semiconductor nanocrystal and capable of linking to an affinity molecule.
 2. The compound of claim 1, wherein said first semiconductor material is a II-VI semiconductor or a III-V semiconductor.
 3. The compound of claim 2, wherein said first semiconductor material is a II-VI semiconductor.
 4. The compound of claim 2, wherein said first semiconductor material is a III-V semiconductor.
 5. The compound of claim 3, wherein said first semiconductor material is MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, or HgTe.
 6. The compound of claim 4, wherein said first semiconductor material is GaAs, InGaAs, InP, or InAs.
 7. The compound of claim 1, wherein said second semiconductor material is a II-VI semiconductor or a III-V semiconductor.
 8. The compound of claim 7, wherein said second semiconductor material is a II-VI semiconductor.
 9. The compound of claim 7, wherein said second semiconductor material is a III-V semiconductor.
 10. The compound of claim 8, wherein said second semiconductor material is MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, or HgTe.
 11. The compound of claim 9, wherein said second semiconductor material is GaAs, InGaAs, InP, or InAs.
 12. The compound of claim 1, wherein said first semiconductor material is CdSe and the second semiconductor material is ZnS.
 13. The compound of claim 1, wherein said linking agent comprises a thiol moiety.
 14. The compound of claim 13, wherein said linking agent further comprises an alkyl group.
 15. The compound of claim 14, wherein said alkyl group is a propyl group.
 16. The compound of claim 1, wherein said linking agent is N-(3-aminopropyl)-3-mercapto-benzamide, 3-aminopropyl-trimethoxysilane, 3-mercaptopropyl-trimethoxysilane, 3-maleimidopropyl-trimethoxysilane, or 3-hydrazidopropyl-trimethoxysilane.
 17. The compound of claim 1, wherein said nanocrystal compound further comprises a glass coating on said shell.
 18. The compound of claim 17, wherein said glass coating comprises a polymeric oxide.
 19. The compound of claim 18, wherein said polymeric oxide is an oxide of silicon, an oxide of boron, an oxide of phosphorus, or a mixture thereof.
 20. The compound of claim 18, wherein said glass coating further comprises a metal silicate, a metal borate or a metal phosphate.
 21. The compound of claim 7, wherein said linking agent is N-(3-aminopropyl)-3-mercapto-benzamide, 3-aminopropyl-trimethoxysilane, 3-mercaptopropyl-trimethoxysilane, 3-maleimidopropyl-trimethoxysilane, or 3-hydrazidopropyl-trimethoxysilane.
 22. The compound of claim 1, wherein said shell epitaxially surrounds said core.
 23. A luminescent semiconductor nanocrystal compound, comprising: a) a water-soluble luminescent semiconductor nanocrystal comprising: i) a core comprising a first semiconductor material; and ii) a core-overcoating shell comprising a second semiconductor material; and b) a linking agent linked to said water-soluble luminescent semiconductor nanocrystal and capable of linking to an affinity molecule.
 24. A luminescent semiconductor nanocrystal compound, comprising: a) a water-soluble luminescent semiconductor nanocrystal comprising: i) a core comprising a first luminescent semiconductor nanocrystal material; and ii) a core-overcoating shell comprising a second semiconductor material; and b) a linking agent linked to said water-soluble luminescent semiconductor nanocrystal and capable of linking to an affinity molecule.
 25. A luminescent semiconductor nanocrystal compound, comprising: a) a water-soluble luminescent semiconductor nanocrystal comprising: i) a core comprising a first semiconductor material; and ii) a core-overcoating shell comprising a second luminescent semiconductor nanocrystal material: and b) a linking agent lined to said water-soluble luminescent semiconductor nanocrystal and capable of linking to an affinity molecule.
 26. A luminescent semiconductor nanocrystal compound, comprising: a) a water-soluble luminescent semiconductor nanocrystal comprising: i) a core comprising a first luminescent semiconductor nanocrystal material; and ii) a core-overcoating shell comprising a second luminescent semiconductor nanocrystal material; and b) a linking agent linked to said water-soluble luminescent semiconductor nanocrystal and capable of linking to an affinity molecule. 